CN1429623A - Method for treating endothelium wond - Google Patents

Abstract

The use of human erythropoietin (EPO) to prevent or treat endothelial injury due to chemotherapy, radiation therapy, mechanical trauma, or to a disease state which damages the endothelium (such as inflammation, heart disease or cancer) is described. The use of EPO in conjuction with the administration of chemotherapeutic agents is described.

Description

Methods of treating endothelial wounds

This application is a divisional application of No. 97199338.6 patent application filed on September 10, 1997, with the title of invention "Method for Treating Endothelial Wounds".

field of invention

The present invention relates to the use of human erythropoietin (EPO) in preventing or treating endothelial trauma caused by chemotherapy, radiotherapy, mechanical trauma or diseases that damage endothelium (such as inflammation, heart disease or cancer). The invention further relates to the use of EPO in combination with chemotherapy.

Background technology of the invention

Erythropoietin (EPO), a glycoprotein produced in the kidney, is the primary hormone responsible for stimulating the production of red blood cells (erythropoiesis). EPO stimulates division and differentiation of committed erythroid progenitors in the bone marrow. Normal plasma erythropoietin levels vary from 0.01-0.03 units/mL and can increase up to 100-1000-fold during periods of hypoxia or anemia. See Graber and Krantz, Annals of Medicine (Ann. Rev. Med. 29: 51 (1978); Eschbach and Adamson Kidney Intl. 28: 1 (1985). Recombinant human erythropoietin (rHuEpo or epoetin alpha ) is commercially available in the form of Epogen® (Amgen Corporation, Thousand Oaks, CA) and Procrit@ (Ortho Biotech, Raritan, NJ). EPO is used to treat anemia, including in combination with cancer chemotherapy, chronic failure, malignancy, Zidovudine treatment-associated anemia in adults and children with rheumatoid arthritis, hemoglobin synthesis disorders, precocious puberty, and HIV infection.

The vascular endothelium is a layer of cells that adheres to the inner vessel wall and is in direct contact with the blood, providing a living natural barrier between the circulatory system and the extravascular space. The endothelium is involved in the transmission of signals and information at the cellular, tissue, and organ levels, and plays a role in both cell-mediated and humoral immune responses. Endothelial cells are metabolically active and normally produce many substances that act on the lumen of blood vessels as well as on platelets. Endothelial vasodilators include prostacyclin ( _PGI2 ) and endothelium-derived relaxing factor (EDRF, which may be nitric oxide or its more stable adducts); these two substances also act to inhibit platelet aggregation.

Trauma or destruction of the endothelium by physical trauma or by processes such as atherosclerotic plaque formation can reduce EDRF production, resulting in vasoconstriction. More diffuse and acute endothelial trauma, such as that caused by chronic hypertension or blood refill after ischemia, also leads to the generation of altered EDRF. Endothelial products localized on the luminal endothelial surface include ectoADPase and thrombomodulin. Vasoconstrictors released from the endothelium include endothelin. Endothelial cells also secrete growth factors that enhance endothelial mitosis and can induce the formation of new blood vessels (angiogenesis). Granulocyte macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF) have been reported to stimulate proliferation and migration of endothelial cells. Interleukin-3 (IL-3) also enhances the proliferation of these cells. See Bussolino et al. Nature 337:471 (1989); Brizzi et al. J. Clin. Invest. 91:2887 (1993).

Summary of the invention

A first aspect of the invention is a method of reducing endothelial trauma caused by a chemotherapeutic agent by administering an endothelium-protecting amount of erythropoietin in combination with the chemotherapeutic agent. The endothelial-protecting amount of erythropoietin can be administered at the same time as the chemotherapeutic agent or before or after administration of the chemotherapeutic agent.

A second aspect of the invention is a method of enhancing endothelial cell suppression in a subject treated with a chemotherapeutic agent comprising administering the chemotherapeutic agent in combination with an endothelium suppressing amount of erythropoietin. The endothelium-inhibiting amount of erythropoietin can be administered at the same time as the chemotherapeutic agent or before or after administration of the chemotherapeutic agent.

Another aspect of the invention is a method of treating a solid hemangioma comprising administering an endothelium-inhibiting amount of erythropoietin in combination with an anticancer chemotherapeutic agent. The endothelium-inhibiting amount of erythropoietin can be administered at the same time as the chemotherapeutic agent or before or after administration of the chemotherapeutic agent.

Another aspect of the invention is a method of treating endothelial trauma resulting from mechanical trauma, exposure to radiation, inflammation, heart disease, or cancer comprising administering to an individual in need of such treatment an endothelium-protecting amount of erythropoietin .

The foregoing and other objects of the present invention will be explained in detail in the specification provided below.

Brief description of the drawings

Figure 1 is a dose response curve showing endothelial cell survival after exposure to cisplatin.

Figure 2 shows the response of endothelial cell cultures simultaneously exposed to cisplatin and different doses of EPO compared to control endothelial cell cultures exposed to cisplatin alone.

Figure 3 shows the response of endothelial cell cultures exposed first to cisplatin and then two hours later to different doses of EPO (compared to control endothelial cell cultures exposed to cisplatin alone).

Figure 4 shows the response of endothelial cell cultures exposed to different doses of EPO followed by cisplatin two hours later (compared to control endothelial cell cultures exposed to cisplatin alone).

Detailed description of the invention

The present inventors have previously demonstrated that recombinant human erythropoietin (EPO) has mitogenic and chemoattractant effects (migration) on human umbilical vein endothelial cells and bovine capillary endothelial cells. See Anagnostou et al. Proc. Natl. Acad. Sci. USA 87:5978 (1990). Migration and proliferation of endothelial cells are key steps in the process of angiogenesis.

The present inventors have found that EPO is effective in preventing and/or repairing endothelial trauma caused by chemotherapeutic agents. The present inventors found that co-administration of EPO with chemotherapeutic agents produced a biphasic response: A dose of EPO protected endothelial cells from the deleterious effects of the chemotherapeutic agent, while increasing doses enhanced the endothelial growth inhibition induced by the chemotherapeutic agent.

The use of EPO to enhance endothelial growth inhibition during chemotherapy may be useful in the treatment of angiogenic tumors, in the hope of preventing or slowing the formation of new blood vessels that support tumor growth. Tumors require an adequate blood supply, and angiogenic factors secreted by tumor tissue stimulate the growth of new blood vessels in the tumor entity. In animal models, inhibition of angiogenic triggers in tumor tissue has been shown to lead to tumor regression. Highly vascularized solid tumors include hemangioblastoma of the cerebellum, ductal breast carcinoma, and squamous cell carcinoma of the larynx. Aberrant angiogenesis is associated with other pathological diseases, including diabetic retinopathy, neovascular glaucoma, rheumatoid arthritis, and psoriasis. The ability of EPO to reduce or prevent abnormal angiogenesis would be useful in preventing or reducing angiogenesis associated with these conditions.

One method according to the invention is the use of EPO as an adjuvant in the chemotherapy of neoplastic diseases. When it is desired to protect the endothelium from the adverse effects of chemotherapeutic agents, EPO is provided in endothelial protective amounts. A second method according to the invention is the use of EPO as an adjuvant in the chemotherapy of neoplastic diseases, where it is desired to enhance the adverse effects of the chemotherapeutic agent on the endothelium (for example to enhance the inhibition of endothelial growth). In this case, EPO was provided in an endothelial inhibiting amount.

As used herein, an endothelial protective amount of EPO refers to an amount that reduces or prevents inhibition of endothelial growth that would otherwise result from exposure to chemotherapeutic agents or radiation, mechanical trauma, or conditions known to traumatize the endothelium. On the other hand, endothelial protective amounts of EPO can be defined as those doses that increase the number of viable endothelial cells after exposure to chemotherapeutic agents or radiation, mechanical trauma, or diseases known to traumatize the endothelium; the increase in the number of viable cells is Refers to an increase compared to the number expected without EPO. The most effective endothelial protective amount of EPO may vary with the time of administration and the etiology of endothelial trauma.

When endothelial trauma is due to exposure to chemotherapeutic agents, the amount of EPO at which EPO is most effective for preserving endothelium will vary depending on whether EPO is administered at the same time as, or before or after, the chemotherapeutic agent and may vary with the specific chemotherapeutic agent used And change.

As used herein, an endothelial inhibiting amount of EPO refers to an amount that enhances or increases the inhibition of endothelial growth that would otherwise result from exposure to chemotherapeutic agents or radiation, mechanical trauma, or disease known to traumatize the endothelium. On the other hand, endothelial-inhibiting amounts of EPO can be defined as those doses that reduce the number of viable endothelial cells following exposure to chemotherapeutic agents or radiation, mechanical trauma, or conditions known to traumatize the endothelium; a reduction in the number of viable cells means A decrease compared to the number expected without EPO. The most effective endothelial inhibitory amount of EPO can vary with the time of administration and the etiology of endothelial trauma.

When endothelial trauma is due to exposure to chemotherapeutic agents, the amount of EPO that is most effective for endothelial inhibition will vary depending on whether EPO is administered at the same time as, or before or after, the chemotherapeutic agent and can vary with the specific chemotherapeutic agent used change.

Endothelial trauma resulting in a reduction in the total number of viable endothelial cells can be assessed by a reduction in endothelial cell proliferation and/or a reduction in the number of viable endothelial cells. This reduction in the number of viable endothelial cells may also be referred to as endothelial growth inhibition or endothelial cell suppression or inhibition.

As used herein, a method of reducing endothelial trauma in an individual resulting from administration of a chemotherapeutic agent refers to a method of reducing or preventing the reduction in viable endothelial cells that would otherwise result from the administration of a chemotherapeutic agent. As used herein, a method of enhancing endothelial suppression in an individual as a result of administering a chemotherapeutic agent to the individual refers to a method of increasing or enhancing the reduction in viable endothelial cells that would otherwise result from the administration of a chemotherapeutic agent.

Trauma to endothelial cells can also be caused by radiation therapy, mechanical trauma, and by diseases such as inflammation, heart disease (eg, atherosclerosis), and cancer. In atherosclerosis, for example, endothelial trauma or dysfunction leads to a diminished vasodilation response and increased platelet deposition on the arterial wall. Serotensin and thromboxane _A2 released from deposited platelets cause arterial constriction and spasm, increase platelet adhesion and aggregation, and promote atherosclerosis. Formation of new coronary vessels by angiogenic stimulation often ameliorates the consequences of coronary artery blockage. The use of EPO in enhancing endothelial growth and/or repairing or preventing endothelial trauma would make it an effective therapeutic agent for endothelial trauma due to mechanical trauma, radiation therapy or due to diseases that adversely affect the endothelium.

As used herein, human erythropoietin (EPO) refers to the naturally occurring human erythropoietin glycoprotein as well as recombinant human erythropoietin (rHuEpo or epoetin alpha, available as Epogen® (Amgen Corporation, Thousand Oaks, CA) and Procrit® ( Commercially available as Ortho Biotechnology, Ratitan, NJ). Peptide analogs of EPO may also be used in the methods of the invention. As used herein, peptide analogs are those compounds that, although not having the same amino acid sequence as EPO, have a similar three-dimensional structure. In a protein molecule that interacts with a receptor, the interaction occurs at a point on the surface of a stable three-dimensional molecule. Peptides that mimic the requisite surface features of the EPO binding domain can be designed and synthesized by arranging the key binding site residues in an appropriate conformation according to known methods. Molecules having a surface area with substantially the same molecular topology as the EPO binding surface will be able to mimic the interaction of EPO with the EPO receptor. Methods are known for determining the three-dimensional structure of peptides and their analogs, sometimes referred to as 'rational drug design techniques'. See, eg, U.S. Patent No. 4,833,092 to Geysen; U.S. Patent No. 4,859,765 to Nestor; U.S. Patent No. 4,853,871 to Pantoliano; U.S. Patent No. 4,863,857 to Blalock (applicant specifically states that all U.S. patent publications cited herein are incorporated by reference in their entirety).

In the methods of the present invention, peptides that mimic the biological activity of erythropoietin (EPO receptor peptide ligands) can be substituted for EPO. The sequences of these peptides may represent fragments of the full-length EPO protein sequence which are capable of binding to the EPO receptor and activating it. In addition, peptides having sequences that are not similar to EPO are useful in the methods of the invention when these peptides mimic the biological activity of EPO. Wrighton et al. reported the identification and characterization of small molecule peptides that bind to and activate the erythropoietin receptor on the surface of target cells, although these peptide sequences are not similar to the primary structure of EPO (Wrighton et al. Science 273:458 (1996, 7, 26)). (These peptide agonists are represented by a disulfide bonded 14 amino acid cyclic peptide with the smallest consensus sequence identified. Livnah et al., Science 273:464 (1996, 7, 26) describe one such peptidomimetic The structure of the complex with the erythropoietin receptor.

As used herein, the term chemotherapeutic agent refers to a cytotoxic antineoplastic agent, ie, a chemical substance used therapeutically to prevent or reduce the growth of tumor cells, preferentially kill tumor cells or interrupt the cell cycle of rapidly proliferating cells. Chemotherapeutic agents are also known as antineoplastic or cytotoxic agents and are well known in the art. As used herein, chemotherapy includes treatment with a single chemotherapeutic agent or with a combination of chemotherapeutic agents. In individuals in need of treatment, chemotherapy may be combined with surgery or radiation or with other antineoplastic treatments.

Exemplary chemotherapeutic agents are vinca alkaloids, epipodophyllotoxin, anthracycline antibiotics, actinomycin D, plicamycin, (puromycin, gramicidin D, paclitaxel (Taxol®, Bristol Myers Squibb), colchicine, cytochalasin B, emimidine, maytansine, and amsacrine (or "mAMSA"). Vinca alkaloids are described in Goodman and Gilman , Pharmacological Basis of Therapy , 1227-1280 (7th edition, 1985) (hereinafter "Goodman and Gilman"). Examples of vinca alkaloids are vincristine, vinblastine, and vindesine. Epipodophyllotoxins are described in Goodman and Gilman, supra 1280-1281. Examples of epipodophyllotoxins are etoposide, etoposide-n-quinone and teniposide. Anthracycline antibiotics are described in Goodman and Gilman, supra 1283-1285. Anthracycline antibiotics Examples are daunorubicin, doxorubicin, mitoxantrone, and bisantrene. Actinomycin D, also known as dactinomycin, is described in Goodman and Gilman, supra 1281-1283. Pleucamycin , also known as mithramycin, described in Goodman and Gilman, supra 1287-1288. Other chemotherapeutic agents include cisplatin (Platinol®, Bristol Myers Squibb); carboplatin (Paraplatin®, Bristol Myers Squibb); Cyclophosphamide (Cytoxan®, Bristol Myers Squibb); Lomustine [CCNU] (CeeNU®, Bristol Myers Squibb); Myers Squibb); carmustine [BCNU] (BiCNU®, Bristol Myers Squibb).

As is known to those skilled in the art, the method of administration of the chemotherapeutic agent will vary depending on the particular chemotherapeutic agent used. Depending on the chemotherapeutic agent used, the chemotherapeutic agent can be administered, for example, by injection (intravenous, intramuscular, intraperitoneal, subcutaneous, intratumoral, intrapleural) or orally.

As used herein, administration of one compound "in conjunction with" another compound means that the two compounds are administered in close enough time that the presence of one alters the biological effect of the other. The two compounds can be administered simultaneously (simultaneously) or sequentially. Simultaneous administration can be accomplished by mixing the compounds prior to administration, or by administering the compounds at the same time point but at different tissue sites or using different routes of administration.

The phrase "simultaneous administration", "concurrent administration" or "simultaneous administration" as used herein means that the compounds are administered at the same point in time or immediately after each other. In the latter case, the two compounds are administered at a time close enough that the observed results are indistinguishable from those obtained when the compounds were administered at the same time point.

Individuals to be treated by the methods of the present invention include human and animal (eg, dog, cat, cow, horse) individuals, preferably mammalian individuals.

Many chemotherapeutic agents act at specific phases of the cell cycle and are only active on cells that are in the process of dividing. The most chemotherapy-sensitive tumors are those with a high percentage of cells in the process of cell division, including but not limited to breast, liver, brain, lung, and ovarian cancers. Highly vascularized solid tumors are amenable to treatment with endothelium-inhibiting amounts of EPO in combination with chemotherapeutic agents because these tumors depend on angiogenesis to provide an adequate blood supply to growing tumor tissue.

EPO used according to the invention may be administered by any suitable means apparent to those skilled in the art. EPO can be administered systemically (eg, intravenously) or locally (eg, by injection into the tumor, into the tissue immediately surrounding the tumor, or into an anatomical cavity containing the tumor). For example, when endothelial-inhibiting amounts of EPO are used as an adjuvant to chemotherapy, EPO can be administered locally into the tumor (or the tissue immediately surrounding the tumor) where it is desired to prevent angiogenesis. For example, in the case of systemic delivery of chemotherapeutic agents, endothelium-protecting amounts of EPO can be administered systemically by intravenous injection.

The dosage and timing of EPO administered in combination with chemotherapeutic agents will similarly depend on the effect desired. The present inventors found that depending on the time of administration of EPO (simultaneously, before or after the administration of the chemotherapeutic agent) and the dose of the administration, EPO can both protect the endothelium from the growth inhibition of the chemotherapeutic agent and enhance the use of chemotherapeutic agents. inhibitory effect on endothelial growth seen with the drug. It will be apparent to those skilled in the art by routine experimentation how to determine the dosage and timing of EPO administration to achieve the desired effect in combination with a particular chemotherapeutic agent.

There is no established maximum amount of EPO that can be administered in single or multiple doses. Doses up to 1,500 units/kg have been administered over three to four weeks without toxic effects due to EPO itself. See Eschbach et al., Prevention of Chronic Uremia (Friedman et al . eds.), Field and Wood Inc., Philadelphia, pp 148-155 (1989). In this method, EPO is administered in an endothelial protective amount when it is desired to protect the endothelium from endothelial trauma and/or endothelial growth inhibition caused by chemotherapeutic agents. A suitable dose for endothelial protection is from about 100 U/kg to about 200 U/kg. In this method, where it is desired to enhance endothelial trauma and/or endothelial growth inhibition by chemotherapeutic agents, EPO is administered in an endothelial inhibiting amount, which may range from about 750 U/kg to about 2000 U/kg. As noted above, the dosage and timing of the administration of EPO in combination with a chemotherapeutic agent will depend upon the desired effect and the chemotherapeutic agent used.

The following examples are provided to illustrate the invention and should not be considered as limiting them.

Example 1 Materials and Methods Cell Culture Human umbilical vein endothelial cells (HUVECs) were obtained from umbilical cords derived from caesarean section. HUVECs were cultured in 25 cm ² T-flasks covered with 0.5% pigskin gelatin (Sigma Chemical Company, St. Louis, MO) by standard methods. Supplemented with 20% of the prescribed fetal bovine serum (FBS) (Hyclone, Logan, UT), 16 U/ml heparin (Sigma), 50 ug/ml endothelial mitogen derived from bovine hypothalamus (Biomedical Technologies, Stoughton, MA) , Medium 199 with 100 U/ml penicillin and 100 ug/ml streptomycin was used for the growth of HUVECs (Life Technologies, Gaithersburg, MD). As known in the art, endothelial cells are characterized by von Willebrand factor antigen positivity, similar and typical cobblestone morphology, and the presence of Weillebrand-Palade bodies. The protection/inhibition assay uses a colorimetric method to determine the number of metabolically active cells following exposure of endothelial cell cultures to the test substance. This analysis uses the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H - a solution of tetrazolium] (MTS) and the electron coupler phenazine methosulfate (PMS; commercially available from Promega Corporation, Madison, Wisconsin). See Denizot and Lang, J. Immunol. Methods 89:271 (1986); Promega Corporation Technical Bulletins 112, 152 and 169. MTS is bioreduced to formazan by dehydrogenases found in metabolically active cells. The amount of formazan is measured as the absorbance at 490 nm, which is directly proportional to the number of viable cells in the culture.

Log endothelial cells grown in complete (supplemented) M199 medium were harvested. At 80-90% confluence, EC culture monolayers were washed with phosphate-buffered saline (PBS), treated with 0.25% trypsin in 1 mM EDTA for 1-2 minutes, and cells were then suspended in complete medium . Cell number and viability were determined using a hemocytometer and trypan blue staining, respectively. Prepare a cell suspension of 7.22×10 ⁴ cells/ml medium, and distribute 90ul (6.5×10 ³ cells) in each well of a 96-well plate. After overnight incubation at 37° C., 5% CO ₂ , in humidified air, EPO and/or chemotherapeutic agents were added at the concentrations and sequences described in the Examples described below. The plates were then incubated for an additional 24 hours. At the end of the incubation period, as recommended by the manufacturer, 20 ul of freshly prepared mixed MTS/PMS (20:1 ratio) solution was added to each well and the plate was incubated for an additional 1-4 hours. The absorbance of each well at 490 nm was recorded using an ELIAS plate counter. LD50 and the effect of various treatments on cell viability and chemosensitivity were determined by plotting the corrected absorbance at 490 nm versus the concentration of the spiked substance (EPO, chemotherapeutic agent or mixture thereof). Statistical studies For the protection/inhibition analysis, experiments were repeated three times. All other experiments were performed at least 5 times. The results were averaged and expressed as mean ± SD. The control group for all experiments consisted of 1-2 wells in triplicate treated with one of the following: 1) 1 ug/ml cisplatin; 2) 50 ug/ml cisplatin; 3) 10 or 20 U/ml EPO; 4) 0.6 or 1.2 U/ml EPO.

Thus, for each experiment, 3-6 wells received the above four control treatments (12-24 control wells in total). Additional controls consisting of triplicate wells of untreated cells were also tested.

The mensuration of embodiment 2 cisplatin LD50

96-well plates containing endothelial cells were prepared as described in Example 1 and incubated overnight at 37°C, 5% CO ₂ , in a humidified atmosphere. A 160ug/ml cisplatin solution was prepared, and serial dilutions were added to each well (5ul/well; the concentration varied from 0.03125ug/ml to 4.0ug/ml. Then the plate was incubated for two days (48 hours)), using Example 1 The described MTS/PMS method determines the viability of endothelial cells. Absorbance of each well at 490 nm was recorded using an ELISA plate counter. The corrected absorbance at 490 nm was plotted against the concentration of cisplatin (ug/ml) (Figure 1) to provide a dose-response curve. The concentration of cisplatin required to produce a 50% maximal response (LD50 of cisplatin) was determined to be 0.45 ug/ml.

Based on the above findings, the effect of EPO on endothelial cells was determined using cisplatin at a dose of 1 ug/ml as provided in the Examples below.

Embodiment 3 Cisplatin and the influence of EPO administered simultaneously on endothelial cells

To determine the effect of the combination of EPO and cisplatin on endothelial cells, serial dilutions of EPO were added simultaneously with cisplatin to endothelial cell cultures.

Endothelial cell cultures were prepared as described in Example 1. Cisplatin (final concentration 1 ug/ml) and 5 ul EPO preparations of various concentrations (final EPO concentration varied from 0.15 to 20 U/ml) were added to each test well at the same time. Endothelial cell viability was determined using the MTS/PMS colorimetric assay described in Example 1. Results were compared to control wells (endothelial cells treated with 1 ug/ml cisplatin only, which was taken as baseline and represented as 0% in Figure 2). The results are provided in Figure 2; "Percentage of control group" is the percent change in optical density at 490 nm relative to the control group, such that "0%" indicates that the test well has a similar number of metabolically active cells as the control group, and "50 %" indicates 50% more and "-50%" indicates 50% less metabolically active cells.

As shown in Figure 2, when EPO was added to the cell culture simultaneously with cisplatin, a biphasic response was observed. When EPO was added simultaneously with cisplatin, endothelial cell cultures treated with 0.15-1.25 U/ml EPO were protected from the traumatic effects of cisplatin. When EPO was added simultaneously with cisplatin, an EPO concentration of 0.3 U/ml provided the greatest protection of endothelial cells; the number of surviving cells was about 30% higher than that observed in control cultures treated with cisplatin alone.

As also shown in Figure 2, when EPO was added simultaneously with cisplatin, endothelial cell growth was inhibited in cultures treated with 5-20 U/ml EPO compared to cultures treated with cisplatin alone. Cultures treated with 5 U/ml EPO and 1 ug/ml cisplatin showed a 33% reduction in the number of viable cells compared to control cells exposed to cisplatin alone.

Example 4 Effects of EPO administered after exposure to cisplatin on endothelial cells

In this experiment, serial dilutions of EPO were added to endothelial cell cultures 2 hours after the cultures were exposed to cisplatin.

Cultures of endothelial cells were prepared as described in Example 1. Cisplatin was added to each test well (final concentration of cisplatin was 1 ug/ml); 2 hours later, EPO preparations were added with final concentrations ranging from 0.15-20 U/ml. Endothelial cell viability was determined using the MTS/PMS colorimetric method as described in the Examples. Results were compared to control wells (endothelial cells treated with 1 ug/ml cisplatin only).

The results, presented in Figure 3, show that a biphasic response was observed when EPO was added to the cell cultures following the addition of cisplatin. Endothelial cell cultures treated with 0.15-5 U/ml EPO were protected from the traumatic effects of cisplatin when EPO was added 2 hours after exposure to cisplatin. In endothelial cell cultures treated with 1.25 U/ml EPO after exposure to cisplatin, the number of surviving cells was 34% higher than in the control group. In contrast, in endothelial cell cultures administered 10-20 U/ml EPO 2 hours after exposure to cisplatin, cell viability was lower than that observed in the control group (exposed to cisplatin alone).

Example 5 Effects of EPO administered prior to exposure to cisplatin on endothelial cells

In this experiment, serial dilutions of EPO were added to endothelial cell cultures 2 hours before exposing the cultures to cisplatin.

Cultures of endothelial cells were prepared as described in Example 1. 5ul of EPO preparation with a concentration of 0.15-20U/ml was added to each test well; 2 hours later, cisplatin was added to each test well (5ul of 1ug/ml of cisplatin). Endothelial cell viability was determined using the MTS/PMS colorimetric method as described in the Examples. Results were compared to control wells (cells treated with 1 ug/ml cisplatin only).

The results are presented in Figure 4, showing a reduction in the number of viable endothelial cells following exposure to EPO 2 hours prior to exposure to cisplatin (compared to control cells exposed to cisplatin alone). Cell proliferation and survival were reduced by 81% compared to the control group. Inhibition was dose dependent; EPO concentrations as low as 5 and 2.5 U/ml decreased cell growth by 58% and 48%, respectively, compared to controls.

The foregoing is a description of the invention and should not be considered as a limitation on them. The invention is defined by the following claims, to which equivalents of the claims are to be included.

Claims (11)

CLAIMS 1. Use of erythropoietin for the manufacture of a medicament for administration in combination with a chemotherapeutic agent to enhance endothelial cell suppression in an individual treated with the chemotherapeutic agent. 2. The use according to claim 1, wherein said erythropoietin is administered simultaneously with said chemotherapeutic agent. 3. The use according to claim 1, wherein said erythropoietin is administered before said chemotherapeutic agent. 4. The use according to claim 1, wherein said erythropoietin is administered after said chemotherapeutic agent. 5. The use according to claim 1, wherein the chemotherapeutic agent is cisplatin. 6. The use according to claim 1, wherein said individual suffers from a tumor growth selected from the group consisting of cerebellar hemangioblastoma, mammary duct carcinoma, and laryngeal squamous cell carcinoma. 7. Use of erythropoietin for the manufacture of a medicament for administration in combination with an anticancer chemotherapeutic agent for the treatment of a solid hemangioma in an individual. 8. The use according to claim 7, wherein said erythropoietin is administered simultaneously with a chemotherapeutic agent. 9. The use according to claim 7, wherein said erythropoietin is administered before said chemotherapeutic agent. 10. The use according to claim 7, wherein said erythropoietin is administered after said chemotherapeutic agent. 11. The use of erythropoietin for the manufacture of a medicament for the treatment of endothelial trauma in an individual caused by mechanical trauma, exposure to radiation, inflammation, heart disease or cancer.